In Vivo Antitumor Activity of a Recombinant IL-7/IL-15 Hybrid Cytokine in Mice

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Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running title: In vivo antitumor Activity of a Recombinant IL-7/IL-15

Title: In vivo antitumor Activity of a Recombinant IL-7/IL-15 Hybrid Cytokine in Mice

Yinhong Song1, 2, Yalan Liu1, Rong Hu1, Min Su1, Debra Rood1, and Laijun Lai1, 3

1 Department of Allied Health Sciences, 3 University of Connecticut Stem Cell Institute,

University of Connecticut, Storrs, CT

2 Medical College, Three Gorges University, Yichang, China

Keywords: cancer treatment, cytokines, IL-7, IL-15, mice

Correspondence: Laijun Lai, M.D., Department of Allied Health Sciences, University of

Connecticut, 1390 Storrs Road, Storrs, CT 06269, USA. Phone: (860) 486-6073; Fax: (860)

486-0534; E-mail: [email protected]

Potential Conflict of Interest: The authors declare that they have no conflict of interest.

Financial Support: This work was partly supported by a grant from the Connecticut

Biomedical Research Program (#2011-0145, to L. Lai).

Word count: 4,591; total number of figures and tables: 6

Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Both IL-7 and IL-15 have become important candidate immunomodulators for cancer

treatment. However, IL-7 or IL-15 used alone suffers from shortcomings, such as short serum

half-life and limited antitumor effect. We have cloned and expressed a recombinant (r)

IL-7/IL-15 fusion protein in which IL-7 and IL-15 are linked by a flexible linker. We then

compared the antitumor effect of rIL-7/IL-15 with the individual factors rIL-7 and/or rIL-15.

We show here that rIL-7/IL-15 has a higher antitumor activity than the combination of the

individual factors in both murine B16F10 melanoma and CT-26 colon cancer models. This

was associated with a significant increase in tumor infiltration of T cells, DCs and NK cells

and a decrease in regulatory T cells (Tregs). In addition, rIL-7/IL-15-treated DCs had higher

expression of costimulatory molecules CD80 and CD86. The higher antitumor activity of

rIL-7/IL-15 is likely due to its longer in vivo half-life and different effects on immune cells.

Our results suggest that rIL-7/IL-15 may offer a new tool to enhance antitumor immunity and

treat cancer.

Introduction

Both IL-7 and IL-15 are the common cytokine receptor γ–chain (γc) family cytokines.

IL-7 plays a central role in the development and maintenance of T cells (1-5). The IL-7

receptor (R) consists of two subunits, the IL-7Rα and γc (1-5), the latter also being a

component of the receptors for IL-2, IL-4, IL-9, IL-15 and IL-21. IL-7Rα is expressed by T

cells and DCs, etc. (1-6). Several studies have shown that IL-7 has antitumor activity (7-12).

For example, tumor cell lines that were transfected to produce rIL-7 locally reduced

tumorigenicity in vivo, which was dependent on CD4+ or CD8+ T cells (7-9, 11). Local or

systemic administration of rIL-7 also had antitumor effects (7, 10), especially when rIL-7 was

combined with cancer vaccines (10, 12).

IL-15 induces the differentiation and proliferation of T and NK cells, enhances the

cytolytic activity of CD8+ T cells, and induces the maturation of DCs (13). The IL-15

receptor is composed of a unique α subunit (IL-15Rα), a β subunit (IL-2R/15Rβ) that is

shared with the IL-2 receptor, and the γc. IL-15 can bind to IL-15Rβ via cis- or

trans-presentation by IL-15Rα. IL-15Rβ is expressed by multiple lymphoid populations, such

as T cells, DCs, NK cells, and NKT cells, etc. (14). In vivo administration of IL-15 has

anti-tumor effects in several mouse tumor models (15-22); however, it has been shown that

administration of IL-15 alone is not optimal (13). Various combination strategies have been

explored to increase the efficacy of IL-15 immunotherapy, including coadministration of

other cytokines or inhibitory antibodies against immune-suppression molecules (17-19, 22).

These approaches produced greater anti-tumor responses than did IL-15 alone.

Because both rIL-7 and rIL-15 have a low molecular weight, they undergo a rapid renal

clearance in vivo, thereby having a short plasma half-life (23-26), which diminishes their in

vivo antitumor effects (24, 25). Linking the coding sequences of IL-15 with other proteins has

increased the IL-15’s plasma half-life. Some of the fusion proteins also increase IL-15

signaling, thereby increasing its efficacy (24, 25, 27-29).

We have previously described a single-chain rIL-7/HGFβ hybrid cytokine liking IL-7

and HGFβ by a flexible linker (30). The in vivo half-life of rIL-7/HGFβ was significantly

longer than that of rIL-7 (31). In addition, rIL-7/HGFβ has different effects on immune cells

(30-32), resulting in a higher antitumor activity (33), as compared with the individual factors

rIL-7 and/or rHGFβ. Here we sought to determine whether a single-chain recombinant hybrid

cytokine containing IL-7 and IL-15 might have a higher antitumor effect than the

combination of the individual factors rIL-7 and rIL-15.

Materials and Methods

Animals and cell lines

Murine B16F10 melanoma and CT-26 colon cancer cells were obtained from the American

Type Culture Collection and the National Cancer Institute in 2008. American Type Culture

Collection characterized the cells by using karyotyping and cytochrome c oxidase I testing.

We passaged the cells for less than 4 months before storing them in liquid nitrogen. Before

the cancer cells were injected into mice, they were cultured in Dulbecco Modified Eagle

Medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS at 37℃ and 5% CO2

humidified air. Cell viability was assessed using a trypan blue exclusion dye method. Cells

with a viability ＞95% were resuspended in PBS for injection. BALB/c, C57BL/6 mice,

homozygous C57BL/6 IFN-γ null mice (IFN-γ-/-), and homozygous TNF-α null mice

(TNF-α-/-) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed,

treated, and handled in accordance with protocols approved by the Institutional Animal Care

and Use Committee of the University of Connecticut.

Construction, expression and purification of human single-chain IL-7/IL-15 hybrid

cytokine.

The human IL-7/IL-15 gene was constructed by adapting a protocol we previously used

to construct the human IL-7/HGFα gene (32). Briefly, the human IL-7 cDNA was amplified

with primers A [(that contains a secret sequence (SS)] and B, and the human IL-15 cDNA

with primer C and D (Supplemental Table 1). The PCR products of IL-7 and IL-15 were

combined and subjected to an additional round of PCR with primers A and D. Because

primers B and C contained the linker sequence encoding (Gly4Ser)2, the IL-7 and IL-15 genes

(IL-7/IL-15) were connected by a flexible linker after the overlap extension PCR. The

IL-7/IL-15 gene was then cloned into a pOptiVEC mammalian expression vector (Invitrogen)

(Figure 1A) that was then transfected into Chinese hamster ovary cell-derived DG44 cells

(Invitrogen) to produce rIL-7/IL-15 protein.

rIL-7/IL-15 protein was then purified from the supernatant of the transfected DG44 cells.

Briefly, the supernatant was concentrated by a prep/scale–tangential flow filter cartridge with

10 kDa molecular weight cut-off (Millipore, Bedford, MA) and diafiltered into washing

buffer. The sample was then applied to serially linked columns of CM and DEAE sepharose

(GE Health Care Biosciences, Piscataway, NJ); after washing, the linked columns were

separated. rIL-7/IL-15 protein was eluted from the DEAE column in the washing buffer

containing NaCl gradient, and further purified by a gel filtration column (16/60 Sephacryl

S-100 high resolution, GE). The purified protein was analyzed by SDS-PAGE and Western

blotting using antibodies against human IL-7 and IL-15. For controls, we also cloned and

expressed human IL-7 (32) or IL-15 (using primers E and D) gene individually, and purified

rIL-7 and rIL-15 proteins from the expression system, respectively.

Evaluation of local tumor growth and pulmonary metastasis

To induce localized tumors, 1×105 B16F10 melanoma cells or 2×105 CT-26 colon cancer

cells were injected s. c. into the flank of syngeneic C57BL/6 or BALB/c mice, respectively.

The indicated doses of rIL-7/IL-15, rIL-7, and/or rIL-15 (or PBS) were then injected into the

tumor injection site at 2-day intervals over the indicated time period. Tumor size (volume)

was determined every other day by caliper measurements of the shortest (A) and longest (B)

diameter, using the formula V = (A2B)/2. Some mice were also injected i.p. with 450 µg

anti-IL-7Rα antibody (clone A7R34) (31), 200 µg anti-IL-15Rβ antibody (clone TMβ1, from

Biolegend), or isotype controls (31, 34) 1 day before the cytokine injection.

For in vivo cell depletions (CD8 T cells, CD4 T cells, or NK cells), mice received the

following antibodies via i.p. injections: anti-CD8 (clone 2.43, from BioXCell), 500

µg/injection; anti-CD4 (clone GK1.5), 200 µg/injection; or anti-NK1.1 (clone PK136), 300

µg/injection on days -3, -1, and +4 of the cancer cell injection (35). Depletions were

confirmed by flow cytometry analysis of blood samples.

To induce pulmonary metastases, 1×105 B16F10 melanoma cells were injected into the

tail vein of syngeneic mice, and rIL-7/15, rIL-7 and rIL15 or PBS were injected i.v. at 2-day

intervals from days 2 to 12. The animals were euthanized at the indicated times after tumor

inoculation. Metastatic tumor nodules in the subpleural regions of the lungs were counted

under a dissecting microscope.

Confocal Microscopy

Frozen sections of tumor tissue were prepared as described (36). The sections were

stained with antibodies including PE-conjugated anti-mouse CD11c, APC-conjugated

anti-mouse CD4, and FITC-conjugated anti-mouse CD8 or FITC-conjugated anti-mouse

NK1.1 (BioLegend, or BD Biosciences, San Jose, CA). All of the sections were then

counterstained with 4’, 6’-diamidino-2-phenylindole (DAPI; Sigma) and observed under a

Nikon A1R Spectral Confocal microscope (Nikon, Kanagawa, Japan). A minimum of 6

sections from each tumor tissue were used to evaluate CD4+, CD8+, CD11c+ and NK1.1+

cells.

Flow cytometry

Single-cell suspensions of tumor tissues and spleen cells were stained with one or more

of the following fluorochrome-conjugated antibodies: CD3, CD4, CD8, CD25, FoxP3,

CD11c, CD80, CD86, NK1.1, IFN-γ and TNF-α (BioLegend, or BD Biosciences, San Jose,

CA, or eBioscience, San Diego, CA). For intracellular staining, cells were labeled with cell

surface antibodies, permeabilized with a Cytofix/Cytoperm solution (BD Biosciences), and

then stained with anti-FoxP3, IFN-γ or TNF-α antibody. The samples were analyzed on a

FACSCalibur flow cytometer (BD Biosciences), and data analysis was performed using

FlowJo software (Ashland, OR).

Evaluation of T cells producing IFN-γ or TNF-α

Splenocytes were treated with red blood cells lysis buffer, washed, and cultured with

irradiated CT-26 colon cancer cells (1×105/well) or B16F10 melanoma cells (0.5×105/well)

for 2 days. Suspended cells were collected, stained with antibodies against CD4, CD8, and

IFN-γ or TNF-α, and analyzed by flow cytometry.

ELISA for detection of IL-7, IL-15 and rIL-7/IL-15

The concentration of IL-7, IL-15 and rIL-7/IL-15 in serum was determined by Human

IL-7 ELISA™ Kit (abcam, Cambridge, MA) and IL-15 ELISA Ready-DET-Go kit

(eBioscience) according to the manufacturer’s instructions. The serum half-life of the

cytokines was calculated as described (31).

Limulus Amebocyte Lysate (LAL) assay

The endotoxin level in purified proteins was determined by the endpoint chromogenic

LAL test according to the manufacturer’s instructions (Lonza, Walkersville, MD).

Statistical analysis

Student’s two-tailed unpaired t test was used for comparisons between two groups, and

one-way ANOVA for more than two groups. The results were considered significantly

different at p<0.05.

Results

1. Expression and purification of human single-chain rIL-7/IL-15 protein

We have successfully constructed the human IL-7/IL-15 gene in which the IL-7 and IL-15

cDNAs were connected by a flexible linker (Figure 1A). The IL-7/IL-15 gene was transfected

and expressed in the pOptiVEC/DG44 mammalian expression system, and rIL-7/IL-15

protein was then purified by ion exchanges and gel filtration. As shown in Figure 1B, a

relatively high purity of rIL-7/IL-15 protein was obtained, as determined by Coomassie

blue-stained SDS-PAGE (lane 2). The identity of the protein was verified by Western blot

using IL-7 and IL-15 antibodies (lane 3 and lane 4). The actual molecular weight (MW) of

the rIL-7/IL-15 was higher than the predicted MW, suggesting that the recombinant protein

was glycosylated. The endotoxin level was less than 0.01 EU/ml of 1 µg of purified

rIL-7/IL-15. In vitro assays show that rIL-7/IL-15 had a slightly lower activity than

equimolar amounts of unfused rIL-7 and rIL-15 in stimulating the proliferation of responding

lymphocytes (Supplemental Figure 1), probably because the fusion affects the binding of

rIL-7/IL-15 to its receptors. 9

2. In vivo administration of rIL-7/IL-15 inhibits local tumor growth

To determine the antitumor activity of rIL-7/IL-15, B16F10 melanoma cells were

injected s.c. into the flank of syngeneic C57BL/6 mice. The mice were then injected at the

tumor site with different doses of rIL-7/IL-15 (2.5, 5, 10 and 20 μg/injection) or control

vehicle (PBS) at 2-day intervals from days 2-12. Tumor volumes in each group were

measured over time and compared statistically. As shown in Figure 2A, tumor growth was

inhibited by rIL-7/IL-15 in a dose-responsive manner, with about 60% inhibition at the

2.5-μg level and greater than 80% inhibition at the 20-μg level.

To compare the antitumor effect of rIL-7/IL-15 with its component cytokines, C57BL/6

mice injected with B16F10 melanoma cells were treated with equimolar amounts of

rIL-7/IL-15 (20 μg/injection), rIL-7 (11.6 μg/injection) and/or rIL-15 (8.4 μg/injection),

according to the above schedule. As shown in Figure 2B, although individual factors rIL-7

and/or rIL-15 inhibited local tumor growth, the single-chain rIL-7/IL-15 hybrid cytokine had

significantly higher antitumor activity than rIL-7 and/or rIL-15.

To test whether the anti-tumor effect of rIL-7/IL-15 is mediated by both IL-7 and

IL-15, some of the mice were also injected IL-7 or IL-15 receptor-blocking antibody

(anti-IL-7Rα or anti-IL-15Rβ antibody), or isotype control. As shown in Figure 2C, either

IL-7 or IL-15 receptor-blocking antibody partly abolished the antitumor activity of

rIL-7/IL-15. The data suggest that the anti-tumor effect is IL-7 and IL-15-specific.

To determine if rIL-7/IL-15 has an antitumor effect on other cancers, BALB/c mice

were injected s.c. with murine CT-26 colon cancer cells, then injected at the tumor site with

equimolar amounts of rIL-7/IL-15 (20 μg/injection), rIL-7 (11.6 μg/injection) and/or rIL-15

(8.4 μg/injection) at 2-day intervals from days 2-20. Again, rIL-7/IL-15 had a higher

antitumor activity than rIL-7 and/or rIL-15 (Figure 2D).

To determine whether rIL-7/IL-15, rIL-7 and/or rIL-15 directly affect the growth of

tumor cells, CT-26 colon and B16F10 melanoma cancer cells were cultured in vitro for 2 to 7

days in the presence of 20 to150 ng/ml rIL-7/IL-15, rIL-7 and/or rIL-15, or PBS. The rate of

tumor cell growth was not significantly different at any dose level of rIL-7/IL-15 from those

observed in cultures containing PBS, or rIL-7 and/or rIL-15 (Supplemental Figure 2). Hence,

the mechanism by which rIL-7/IL-15 inhibits the growth of B16F10 melanoma or CT-26

colon cancer in vivo would not appear to involve direct cytotoxic or cytostatic activities.

3. rIL-7/IL-15 induces significant infiltration of T cells, NK cells, NKT cells and DCs,

but decreases infiltration of Tregs into the tumors

To determine the mechanisms by which rIL-7/IL-15 has antitumor activity, we assessed

the percentage of immune cells in tumor tissues. On day 14, after B16F10 cancer cell

inoculation, single-cell suspensions of tumor tissues from mice treated with equimolar

amounts of rIL-7/IL-15, rIL-7 and/or rIL-15 (Figure 2B) were analyzed for CD4+ and CD8+ T

cells, CD4+CD25+Foxp3+ Tregs, CD3-NK1.1+ NK cells, CD3+NK1.1+ NKT cells and CD11c+

DCs by flow cytometry. As shown in Figure 3A and B, rIL-7 increased the tumor infiltration

of both CD8+ and CD4+ T cells, while rIL-15 increased the percentage of CD8+ T cells, but

not that of CD4+ T cells. The combination of rIL-7 and rIL-15 significantly increased the

percentage both CD4+ and CD8+ T cells. However, rIL-7/IL-15 hybrid cytokine had higher

activity than rIL-7 and/or rIL-15 (Figure 3A, B).

Because Tregs can inhibit immune functions and enhance the growth of cancer cells in

vivo (37), we determined whether rIL-7/IL-15 treatment affected the immune suppressive

cells in tumors. rIL-7 and/or rIL-15 did not significantly affect infiltration of

CD4+CD25+Foxp3+ Tregs in the tumors (Figure 3A). In contrast, rIL-7/IL-15 treatment

significantly reduced the percentage of Tregs (Figure 3A).

Similar to the effect on CD8+ T cells, rIL-7 and/or rIL-15 increased tumor infiltration of

NK cells and NKT cells, and rIL-7/IL-15 had a higher activity than the individual factors

rIL-7 and/or rIL-15 on NK cells (Figure 3A, C). However, rIL-7/IL-15 did not have a higher

activity than rIL-7 and rIL-15 on NKT cells (Figure 3A, C).

Because DCs play a critical role in the activation of T cells, we analyzed CD11c+ DCs in

the tumors. As shown in Figure 3A, rIL-7 did not significantly affect tumor infiltration of

DCs. In contrast, rIL-15 increased the percentage of DCs in the tumors. rIL-7/IL-15 had a

higher effect in the tumor infiltration of DCs than rIL-15 used alone or mixed with rIL-7

(Figure 3A). We also examined the expression of CD80 and CD86 on the DCs. Again, rIL-7

did not significantly affect DCs, and rIL-15 increased the expression of CD80 and CD86 on

the DCs. rIL-7/IL-15 treatment resulted in a higher expression of CD80 and CD86 than

rIL-15 used alone or mixed with rIL-7 (Figure 3D), indicating that the DCs had undergone

activation and maturation after rIL-7/IL-15 treatment.

The increased infiltration of CD4+ and CD8+ T cells, DCs, and NK cells in the

rIL-7/IL-15-treated tumors was confirmed by confocal microscopic observations (Figure 3E,

F).

In addition to the tumors themselves, there was a parallel increase in the numbers of

CD4+ and CD8+ T cells, NK cells and activated DCs, and a decrease in the number of Tregs

in the spleen of the rIL-7/IL-15-treated melanoma-bearing mice (Supplemental Figure 3).

To determine whether CD4 and CD8 T cells, and NK cells mediated the antitumor

activity, some of the B16F10 bearing-mice were also injected with anti-CD8, anti-CD4, or

anti-NK1.1 antibody. As shown in Figure 3G, depletion of CD4 or CD8 T cells, or NK cells

partly abrogated the antitumor effect. The data suggest that CD4 and CD8 T cells, and NK

cells mediated the antitumor activity of rIL-7/IL-15.

4. rIL-7/IL-15 induces a tumor-specific immunological response

To determine whether a tumor-specific immunological response has been generated, we

examined the percentage of IFN-γ-producing CD4+ and CD8+ T cells from the spleens of

cytokine-treated tumor-bearing mice after in vitro stimulation with homologous or

heterologous tumor cells. To this end, single cell suspensions from the spleens of day 22,

cytokine-treated, CT-26 colon cancer-bearing mice were stimulated with CT-26 cells. Cells

stimulated with B16F10 melanoma cells served as specificity controls. Two days after the

stimulation, the percentage of IFN-γ or TNF-α producing CD4+ and CD8+ T cells were

analyzed by flow cytometry.

As shown in Figure 4A-C, the percentages of IFN-γ producing CD4+ and CD8+ T cells

among the cultured splenocytes from rIL-7 and/or rIL-15-treated mice were 2.3 to 3.7- and

1.5 to 1.7-fold, respectively, higher than those from PBS-treated mice after stimulation with

CT-26 cells. In contrast, the percentages of IFN-γ producing CD4+ and CD8+ T cells among

the cultured splenocytes from rIL-7/IL-15-treated mice were elevated 8- and 2.3-fold,

respectively. We also analyzed the percentage of TNF-α producing CD4+ and CD8+ T cells

among the cultured splenocytes from the cytokine-treated mice. The percentages of TNF-α

producing CD4+ and CD8+ T cells from rIL-7/IL-15-treated mice were also significantly

higher than those of the T cells from rIL-7 and/or rIL-15-treated mice (Figure 4D-F). These

results suggest rIL-7/IL-15-treatment greatly enhances systemic immunological responses to

tumor-specific antigens in vivo.

To determine whether IFN-γ and TNF-α play a role in the antitumor effects of

rIL-7/IL-15, IFN-γ-/- or TNF-α-/- mice were used as the recipients for B15F10 melanoma cells.

As shown in Figure 4G and 4H, rIL-7/IL-15 treatment resulted in less than 40% tumor

growth inhibition in IFN-γ-/- or TNF-α-/- mice. As compared with more than 85% tumor

inhibition in the wild-type mice (Figure 2B, D), the data suggest that IFN-γ and TNF-α play a

role in the antitumor effect of rIL-7/IL-15.

5. rIL-7/IL-15 inhibits pulmonary metastases of melanoma and colon cancer

Having established that rIL-7/IL-15 inhibited local tumor growth, we wanted to assess

whether rIL-7/IL-15 could also inhibit metastatic tumors. To this end, C57BL6 mice were

injected i.v. with B16F10 melanoma cells to establish pulmonary metastases. The mice were

then treated with equimolar doses of rIL-7/IL-15 (20 μg/injection), rIL-7 (11.6 μg/injection)

plus rIL-15 (8.4 μg/injection), or PBS at 2-day intervals from days 2-12. The mice were

euthanized on day 14 after tumor cell inoculation. The lungs were removed and weighed, and

tumor colonies on the surface of the lung were counted. rIL-7/IL-15 treatment reduced the

numbers of metastatic nodules on the lungs by approximately 8-fold, as compared with

2.7-fold after rIL-7 and rIL-15 treatment (Figure 5). Similar antimetastatic patterns were

observed in the lungs of BALB /c mice after i.v. injection of CT-26 colon cancer cells and

treated with rIL-7/IL-15, or rIL-7 plus rIL-15 (Supplemental Figure 4). These results indicate

that rIL-7/IL-15 also has higher antimetastatic activity than the combination of the individual

factors rIL-7 and rIL-15.

6. rIL-7/IL-15 has a longer serum half-life than rIL-7 and/or rIL-15

It has been shown that both rIL-7 and rIL-15 have a short serum half-life due to their low

molecular weights (23-26). Because rIL-7/IL-15 has a higher molecular weight, we reasoned

that it should have a longer half-life than rIL-7 or rIL-15. Indeed, the serum half-life of

rIL-7/IL-15 was about 6-fold or 18-fold longer than that of rIL-7 or rIL-15, respectively

(Figure 6).

Discussion

Antitumor immunotherapy is designed to stimulate the immune system to reject and

destroy cancers (13). Although either rIL-7 or rIL-15 can enhance antitumor immunity, both

cytokines have a short in vivo half-life, which limit their antitumor activity. We show here

that rIL-7/IL-15 hybrid cytokine has higher antitumor activity than the individual factors

rIL-7 and/or rIL-15 in both melanoma and colon cancer mouse models. This is related to the

higher tumor infiltration of CD4 and CD8 T cells, DCs, NK and NKT cells, the maturation of

DCs, and the inhibition of Tregs induced by rIL-7/IL-15.

It is possible the higher antitumor activity of rIL-7/IL-15 is due to its longer in vivo

half-life, broader activity and different effects on immune cells, as compared with the

individual factors rIL-7 and/or rIL-15. Either rIL-7 or rIL-15 has a low molecular weight and

can undergo a rapid renal clearance in vivo, resulting in a short serum half-life (23-26). The in

vivo half-life of rIL-7 was increased when it was administered in a liposome-encapsulated

form or as an IL-7/anti-IL-7 antibody complex (23, 38). Our previous data had also shown

that the in vivo half-life of another hybrid cytokine rIL-7/HGFβ was 7-fold greater than rIL-7

(31). Similarly, IL-15 fused with other proteins increased its in vivo half-life (24, 25, 27-29).

Consistent with these data, we show here that rIL-7/IL-15 has 6-fold and 18-fold longer in

vivo half-life than rIL-7 and rIL-15, respectively.

Our data also indicate that rIL-7/IL-15 has different effects on immune cells, as

compared with the individual factors. For example, although rIL-7 and/or rIL-15 treatment

did not significantly affect the percentage of Tregs, rIL-7/IL-15 significantly decreased the

percentage or number of Tregs in the tumors and the spleens. Our results are consistent with

previous reports that rIL-7 or rIL-15 alone has little or no effect on Tregs (13, 39-41).

Although the mechanisms by which rIL-7/IL-15 inhibits Tregs remain to be investigated, it is

possible that rIL-7/IL-15 cross-linked both the receptors on Tregs, which resulted in signal

cross-talk downstream of the receptors, leading to novel functional readouts, such as the

inhibition of the survival and/or growth of Tregs.

It has been reported that IL-15 has both paracrine and autocrine actions on DCs, resulting

in the maturation of DCs (41), while IL-7 maintains the immature phenotype of DCs (41, 42).

Our data show that rIL-7 treatment neither significantly affects the percentage of DCs in the

tumors, nor the expression of CD80 and CD86 by the DCs. Although rIL-15 increased both

the intratumor infiltration and the expression of CD80 and CD86 on DCs, rIL-7/IL-15 hybrid

cytokine had a higher activity than rIL-7 and/or rIL-15 on DCs in the tumors and the spleen.

NK cells are innate lymphoid cells involved in the immuno-surveillance of cancers. Our

data show that rIL-15 significantly increased NK cells in the tumors and spleen, and that

rIL-7 modestly increases NK cells. These results are consistent with previous reports that

IL-15 has a central role in the development, homeostasis, and activation of NK cells (13) and

IL-7 only plays a minor role in NK homeostasis (6, 43, 44). Again, rIL-7/IL-15 has a higher

activity than rIL-7 and/or rIL-15 on NK cells. However, although both rIL-7 and rIL-15

increased the percentage or number of NKT cells in the tumors and in the spleen, rIL-7/IL-15

did not have a higher effect than rIL-7 + rIL-15 on NKT cells.

Both IL-7 and IL-15 play an important role in the differentiation, survival and

proliferation of T cells. We have shown that rIL-7 treatment increases the percentages of both

CD4 and CD8 T cells. rIL-15 increases the percentage of CD8 T cells and only modestly

affects CD4 T cells. Although the combination of rIL-7 and rIL-15 had a synergistic effect in

increasing CD4 and CD8 T cells, rIL-7/IL-15 had a higher effect than rIL-7 + rIL-15 on CD4

and CD8 T cells.

The higher activities of rIL-7/IL-15 on DCs, NK and T cells could be due to its longer in

vivo half-life. In addition, it is possible that rIL-7/IL-15 also induced receptor cross-linking

on these cells and subsequent signal cross-talk. This might only happen in DCs, NK and T

cells, but not in NKT cells since rIL-7/IL-15 did not have a significantly higher effect on

NKT cells than rIL-7 + rIL-15.

In summary, although the precise mechanisms by which rIL-7/IL-15 has higher

antitumor effect remain to be further determined, this hybrid cytokine has the potential to be

used in enhancing antitumor immunity, thereby treating a variety of tumors.

References

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FIGURE LEGENDS

Figure 1. Strategy for molecular cloning of IL-7/IL-15 and characterization of purified

rIL-7/IL-15 protein. (A) IL-7, linker, and IL-15 cDNAs were constructed by overlapping PCR.

IL-7/IL-15 cDNA was then ligated into the pOptiVEC vector. (B) Lane 1) MW markers; L2)

Coomassie blue-stained SDS-PAGE; L3) Western blot with anti-human IL-7 antibody; L4)

Western blot with anti-human IL-15 antibody.

Figure 2. rIL-7/IL-15 inhibits the growth of localized melanoma and colon cancer. (A-C)

C57BL/6 mice were injected s.c. with 1×105 B16F10 melanoma cells followed by

intratumoral injections with (A) rIL-7/IL-15 (2.5, 5, 10, or 20 mg) or PBS, (B) equimolar

doses of rIL-7/IL-15 (20 μg), rIL-7 (11.6 µg) and/or rIL-15 (8.4 µg) or PBS at 2-day intervals

between days 2 to 12 after tumor inoculation, or (C) anti-IL-7Rα antibody, anti-IL-15Rβ

antibody, or isotype control on day 1, and rIL-7/IL-15 (20 μg) or PBS at 2-day intervals

between days 2 to 12 after tumor inoculation. (D) BALB/c mice were injected s.c. with 2×105

CT-26 colon cancer cells, followed by intratumoral injections with rIL-7/IL-15 (20 μg), rIL-7

(11.6 µg) and/or rIL-15 (8.4 µg) or PBS at 2-day intervals between days 2 to 20 after tumor

inoculation. Tumors were measured every other day. The mean tumor volume (mm3) ± SD at

the indicated time points is shown. These data are representative of 2 independent

experiments with 3-5 mice per group with similar results. 22

Figure 3. Treatment of localized tumors with rIL-7/15 increases the percentages of CD4+ and

CD8+ T cells, NK, NKT cells, and DCs, but decreases the percentages of Tregs in the tumors.

(A-F) C57BL/6 were injected s.c. with B16F10 melanoma cells and treated with equimolar

doses of rIL-7/IL-15, rIL-7 and/or rIL-15 as in Figure 2B. Fourteen days after tumor

inoculation, the tumors were harvested. (A-D) Single-cell suspensions of tumor tissues were

analyzed for immune cells by flow cytometry. (A) The percentages of CD4+ and CD8+ T cells,

CD4+CD25+Foxp3+ Tregs, CD3-NK1.1+ NK cells, CD3+NK1.1+ NKT cells, and CD11+ DCs.

(B, C) Representative flow cytometric profiles showing the percentage of (B) CD4+CD8- and

CD4-CD8+ T cells and (C) CD3-NK1.1+ NK cells and CD3+NK1.1+ NKT cells in the

rIL-7/IL-15-treated tumors. (D) Relative fluorescence intensity (MFI) of CD80 and CD86 on

the DCs. The mean ± SD is shown. These data are representative of 2 independent

experiments with 5 mice per group with similar results. * P < 0.05 as compared with the

PBS-treated group; ** P < 0.05 as compared with the rIL-7 + rIL-15-treated groups. (E, F)

Tumor sections were analyzed for presence of (E) CD11c+ cells, CD4+ T cells, CD8+ T cells

and (F) NK1.1+ cells by immunofluorescence. (E) DAPI (blue), CD11c+ (red), CD4+ (yellow),

and CD8+ (green). (F) DAPI (blue) and NK1.1+ (green). Original magnification: 200×.

Representative tumor sections are shown. (G) C57BL/6 were injected s.c. with B16F10

melanoma cells and treated with rIL-7/IL-15 or PBS as in Figure 2B. The mice were also

injected i.p. with anti-CD8, anti-CD4, or anti-NK1.1 on days -3, -1, and +4 of the cancer cell

injection. The mean tumor volume (mm3) ± SD at the indicated time points is shown. These

data are representative of 2 independent experiments with 3-5 mice per group with similar

results.

Figure 4. rIL-7/IL-15 treatment results in tumor specific T cell responses. (A-F) BALB/c

mice were injected s.c. with CT-26 colon cancer cells and treated with equimolar doses of

rIL-7/IL-15, rIL-7 and/or rIL-15 as in Figure 2D. Twenty-two days after tumor inoculation,

splenocytes were cocultured with irradiated CT-26 colon cancer cells or B16F10 melanoma

cells at 37 ℃ for 2 days. CD4+ and CD8+ T cells that produce IFN-γ- or TNF-α were analyzed

by flow cytometry. (A) Representative flow cytometric profiles showing the percentage of T

cells in splenocytes and the percentage of IFN-γ-producing CD4+ and CD8+ T cells. Mean +

SD of the percentage of IFN-γ-producing (B) CD4+ and (C) CD8+ T cells. (D) Representative

flow cytometric profiles showing the percentage of T cells in splenocytes and the percentage

of TNF-α-producing CD4+ and CD8+ T cells. Mean + SD of the percentage of

TNF-α-producing (E) CD4+ and (F) CD8+ T-cells. (G, H) IFN-γ-/- or TNF-α-/- and their

wild-type mice were injected s.c. with B16F10 melanoma cells and treated with rIL-7/IL-15

or PBS as in Figure 2B. The mean tumor volume (mm3) ± SD at the indicated time points is

shown. These data are representative of 2 independent experiments with 3-5 mice per group

with similar results. *P<0.05 or **P < 0.01 as compared with the B16F10-treated groups.

#P<0.05 as compared with the rIL-7 + rIL-15-treated groups.

Figure 5. rIL-7/15 inhibits the formation of pulmonary metastases by melanoma cells.

C57BL/6 mice were injected i.v. with 1×105 B16F10 cells, followed by i.v. injections with

equimolar doses of rIL-7/IL-15 (20 µg), rIL-7 (11.6 µg) and rIL-15 (8.4 µg) or PBS at 2-day

intervals between days 2 and 12. The mice were euthanized on day 14 after tumor cell

inoculation. The total tumor nodules visible at the surface of the lungs were counted under a

dissecting microscope. These data are representative of 2 independent experiments, with 5

mice per group, ** P < 0.01.

Figure 6. rIL-7/IL-15 has a longer serum half-life than rIL-7 or rIL-15. Mice were injected

i.p. with optimal equimolar amounts of rIL-7/IL-15 (20 µg), rIL-7 (11.6 µg), or rIL-15 (8.4

µg), and then bred over time (0.25, 0.5, 1, 2, 4, 8, 16, 22, 44, 80, and 120 hours after

treatment). The concentration of rIL-7/IL-15, rIL-7 and rIL-15 in mouse serum was

examined by IL-7- or IL-15-specific ELISA. These data are representative of 2 independent

experiments with 3 mice per group.

In vivo antitumor Activity of a Recombinant IL-7/IL-15 Hybrid Cytokine in Mice

Yinhong Song, Yalan Liu, Rong Hu, et al.

Mol Cancer Ther Published OnlineFirst July 29, 2016.

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